Production of exopolysaccharides unattached to the surface of bacterial cells

ABSTRACT

The present invention is directed to a  Sphingomonas  bacteria and a method of producing exopolysaccharides by culturing a  Shpingomonas  bacteria in a fermentation broth for a time and temperature effective for providing a sphingan exopolysaccharide in a slime form.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/905,829, filed Jul. 13, 2001 now U.S. Pat. No. 6,605,461, nowallowed, which application claims the benefit of U.S. Provisional PatentApplication No. 60/220,145, filed Jul. 24, 2000, wherein theseapplications are incorporated herein by reference in their entireties.

The present invention relates to the production of exopolysaccharidesand bacteria for the production of exopolysaccharides. Moreparticularly, the bacteria of the present invention produceexopolysaccharides in a slime form that is unattached to the surface ofthe bacterial cell.

BACKGROUND

There is an ever increasing demand for inexpensive and environmentallyacceptable viscosifiers, bioemulsifiers and biodegradable polymers.Exopolysaccharides are an example of compounds that are useful for thesepurposes because of their distinctive rheological properties.Exopolysaccharides, such as for example gellan, welan and rhamsan, areproduced commercially for applications in foods, cosmetics, and inoil-field production, and for other applications. Each exopolysaccharidedisplays a different characteristic set of aqueous rheologicalproperties including resistance to shear, compatibility with variousionic compounds, and stability to extreme temperatures, pH and saltconcentrations.

Exopolysaccharides can be produced through bacterial fermentations.Different strains of the genus Sphingomonas produce exopolysaccharidesincluding gellan, welan, rhamsan, S-88, S-7, S-198, NW11, and S-657, toname some examples (Pollock 1993, J. Gen. Microbiol. 139:1939-1945).There are many other exopolysaccharides made by other strains ofSphingomonas bacteria. The exopolysaccharides produced by Shpingomonasbacteria are referred to as “sphingans” with reference to the commongenus as a source. At least three sphingans (gellan, welan, and rhamsan)are produced commercially by large scale submerged fermentation.

The biotechnology industry has responded to the demand forexopolysaccharide compounds by increasing the availability of a varietyof bacterial exopolysaccharide products that are acceptable forcommercial use. Although many of the bacterial exopolysaccharideproducts offer a wide range of attractive improvements oversynthetically produced materials, they remain relatively expensive toproduce. The expense is generally associated with costs of recovery andpurification of the desired product.

Higher fermentation yields of exopolysaccharides have occurred as aresult of improvements and alterations of bacterial strains, and betterunderstanding of bacterial biosynthesis and optimization of fermentationconditions. This satisfies one of the important steps in recoveringadequate amounts of the polymer for potential industrial applications.However, increased exopolysaccharide concentration in the fermentationprocess results in increased viscosities which require higher inputs ofenergy to effectively disperse oxygen and nutrients in the fermentationbroth. Hence, fermentations that provide higher exopolysaccharide yieldshave resulted in correspondingly higher production costs.

Recovery of exopolysaccharides remains a difficult and costly step.Bacterial strains from the genus Shpingomonas produce exopolysaccharideswhich remain attached to the cell surface (Pollock et al. 1999, J.Indust. Microciol. Biotechnol. 23: 436-441). The attached polymers forma capsule around the bacteria. The capsule of polysaccharide is notreadily separated from the bacteria. Even after diluting a fermentationbroth with sufficient water to reduce the viscosity, the capsule remainsattached to the bacterial cells and the cells cannot be separated fromthe capsule by centrifugal sedimentation. Other physical or chemicalmethods are required to separate the cells from the capsule. Forexample, partial hydrolysis of the polysaccharides with acid can be usedto release most of the polysaccharide from the cells by randomlybreaking the polymer chains near to the point of attachment to the cell.

Recovery of exopolysaccharide, regardless of the conditions used toproduce it, typically involves a precipitation step. The precipitatedexopolysaccharide is then recovered by centrifugation. A typical methodfor recovering gellan and welan gums is a follows. Immediately afterfermentation the culture broths are heated to at least 90° C. to killthe living bacteria. Both gums are then separated from the culture brothby precipitation with approximately 2 volumes of isopropylalcohol, andthe precipitated polysaccharide fibers are collected, pressed, dried andmilled. The alcohol is recovered by distillation. In this most simpleprocess the polysaccharide remains attached to the cells, such that whenthe dried and milled polysaccharides is resuspended in water thesolution is not transparent. In the case of gellan gum additional stepscan be introduced to purify the polysaccharide away from the bacterialcells so that the resuspended product is more transparent. Before thealcohol precipitation, the culture broth is centrifuged or filtered orboth while the temperature is maintained above the critical transitiontemperature between a highly viscous state and a liquefied state whichis amenable to centrifugation or filtration. These processes aredisclosed in U.S. Pat. No. 4,326,052 (gellan); U.S. Pat. No. 4,326,053(gellan); U.S. Pat. No. 4,342,866 (welan); U.S. Pat. No. 3,960,832(S-7); and U.S. Pat. No. 4,535,153 (S-88), which are hereby incorporatedby reference.

A major inefficiency associated with a typical product recovery protocolis incomplete recovery of the exopolysaccharide. Bacterialexopolysaccharides are attached to the producing cells with varyingdegrees of tenacity. Those bacteria that have relatively securelyattached exopolysaccharides are less likely to shed them into themedium, thus reducing the amount of exopolysaccharide available forrecovery in the precipitation step and increasing the process stepsneeded to separate the exopolysaccharides from the producing cells.

Advantages, features and characteristics of the present invention willbecome more apparent upon consideration of the following description andthe appended claims.

SUMMARY

The present invention is directed to a method of producing sphinganexopolysaccharides and Shpingomonas bacteria that produce the sphinganexopolysaccharides. The present invention provides a method where thesphingan exopolysaccharides are produced in a slime form such that thesphingan exopolysaccharide is unattached to the surface of theShpingomonas bacteria. Production of the sphingan exopolysaccharide in aslime form requires less energy as compared to fermentations where theexopolysaccharide remains attached to the bacterial cell in a capsuleform. Further, separation and recovery of sphingan exopolysaccharideproduced in a slime form is more efficient as compared to recovery ofsphingan exopolysaccharide produced in a capsule form.

In one aspect, the present invention provides a method for producingsphingan exopolysaccharides that includes culturing Shpingomonasbacteria in a fermentation broth for a time and temperature effectivefor providing a sphingan exopolysaccharide in a slime form. In animportant aspect of the invention, Shpingomonas bacteria that have beengenetically mutated to produce a slime form of sphinganexopolysaccharide, are utilized in a submerged fermentation. Examples ofgenetically mutated Shpingomonas bacteria include ATCC PTA-3487 (strainX287), ATCC PTA-3486 (strain X530), ATCC PTA-3485 (strain Z473), ATCCPTA-3488(strain X031), and mixtures thereof.

Fermentation of the genetically mutated Shpingomonas bacteria of thepresent invention provides a fermentation broth that includes theexopolysaccharide in a slime form. The viscosity of the fermentationbroth is dependent on the amount of sphingan exopolysaccharide that isproduced, which is in turn dependent on the amount of sugar convertedduring the fermentation into exopolysaccharide. If either the sugarconcentration at the beginning of the fermentation or the efficiency ofconversion of sugar into exopolysaccharide is increased, then theviscosity of the broth will increase correspondingly. For example, afermentation with Shpingomonas strain X287 of about 48 to about 96 hoursat a temperature of about 25° C. to about 35° C., results in theproduction of gellan gum in the slime form. The resulting fermentationbroth has a viscosity of about 15,000 to about 30,000 cp, preferablyabout 15,000 to about 20,000 cp, and from about 20 to about 25 g/L astotal biomass. Generally, one-half to three-quarters of this biomassrepresents the exopolysaccharide itself.

In this aspect of the invention, fermentation broths produced throughthe fermentation of genetically mutated Shpingomonas bacteria of thepresent invention have a viscosity of less than about 20,000 cp,whereas, fermentation broths produced through the fermentation ofcorresponding parental strains have a viscosity of more than about40,000 cp. The reduced viscosity provided by the slime form of theexopolysaccharide results in more efficient mixing and aeration, andlower energy consumption.

Mixing, aeration and energy consumption during fermentation aredependent on the size and shape of the fermentation vessel, the type,size and quantity of the fermentation vessel impellers, and the cultureviscosity. For example, with identical fermentation equipment andoperating conditions, a wild type parental strain can be grown tostationary phase, which for Shpingomonas bacteria is an absorbance at600 nm exceeding about 15. At stationary phase, the fermentation brothwill have no detectable oxygen as measured by oxygen sensors inside thevessel. By contrast, the fermentation broth for a genetically mutatedbacteria of the present invention grown to stationary phase will have adissolved oxygen level of at least about 5 percent saturation of water.In addition, maintenance of a positive oxygen level during the entireduration of a fermentation with a genetically mutated bacteria of thepresent invention leads to higher rates of production ofexopolysaccharides.

Another example of the advantages of producing exopolysaccharide in aless viscous slime form is the possibility of adjusting the pH withinthe vessel by addition of exogenous acid or base. When the viscosityexceeds about 20,000 cp, as in a fermentation with a parental strain,rapid mixing of acid or base to produce a homogenous solution is notpossible. Instead, regions of high concentrations of acid or base arecreated. Respectively, the concentrated acid or base can hydrolyze thepolysaccharide or cleave acyl groups from the polysaccharide. Efficientcontrol of pH during the entire duration of a fermentation can alsoincrease productivity.

In an important aspect, the present invention is effective for providinga fermentation broth that results in improved downstream recovery andprocessing of sphingan exopolysaccharides as compared to fermentationbroths where sphingan exopolysaccharides are produced in a capsule form.In this aspect of the invention, sphingan exopolysaccharides aretypically recovered by alcohol precipitation. Volume requirements foralcohol precipitation may be reduced from about 2 volumes to about 1 toabout 1.5 volumes per volume of fermentation broth. Further,temperatures for effective precipitation may be reduced from about 90°C. to about 25° C. to about 50° C.

After fermentation, and prior to precipitation with alcohol, thefermentation broth containing exopolysaccharides may be treated byadditional processing steps. For example, to kill bacterial cells, thebroth temperature may be raised to greater than 80° C. for at least 15minutes. Acyl groups may be removed from the polysaccharide by addingalkali to the heated broth to achieve a pH of about 9 or higher. In thespecial case of gellan gum, these two steps cause the fermentation brothviscosity to decrease sufficiently to allow separation of the bacterialcells from the polysaccharide by physical means such as by eitherfiltration or sedimentation by centrifugation, or a combination of thetwo processes. However, when gellan gum is produced in a capsule form,the separation steps must be accomplished while the fermentation brothtemperature is maintained above 80° C. At temperatures below 80° C.,gellan gum in the culture broth will solidify into a firm gel with thechains of the gellan crosslinked by divalent cations in the culturemedium. If the gellan gum forms a gel, it becomes impossible to recoverthe gellan by precipitation, and the gelled material must be discarded.By contrast, when the exopolysaccharide is in the slime form, viscosityis reduced and the temperature required to prevent formation of a gel isreduced. In this aspect of the invention, the temperature during thecell-separation step can be reduced to as low as about 60° C.

In accordance with this aspect of the invention, the process yieldsabout 10 to about 20 grams expolysaccharide per liter of broth. Thepurity typically exceeds at least about 80%.

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al. (1994)Dictionary of Microbiology and Molecular Biology, second edition, JohnWiley and Sons (New York) provides one of skill with a generaldictionary of many of the terms used in this invention. All patents andpublications referred to herein are incorporated by reference herein.For purposes of the present invention, the following terms are definedbelow.

The terms “sphingan” and “sphingan exopolysaccharide” as used hereinrefer to a group of related but distinct exopolysaccharides secreted bymembers of the genus Shpingomonas (Pollock, J. Gen. Microbiology139:1939-1945, 1993). The structures of the sphingans are all somewhatrelated. The main chain of each sphingan consists of a related sequenceof four sugars: D-glucose, D-glucuronic acid, L-mannose and L-rhamnose.Polysaccharide members of the sphingan group are distinguishable fromeach other by virtue of the carbohydrates which comprise the polymerbackbone and the sidechains. The sphingan polysaccharides may containcarbohydrate side chains and acetyl or pyruvyl groups attached tocarbohydrates on the polymer backbone. See Mikolajczak, et al., Appl.and Env. Microbiol., 60:402, (1994). Gellan, welan, rhamsan, S-88, S-7,NW-11, S-198 and S-657 are examples of exopolysaccharides in thesphingan group.

Typically, members of the sphingan exopolysaccharide family may berepresented by the following general repeating chemical structure:

wherein Glc is glucose; GlcA is glucuronic acid or 2-deoxy-glucuronicacid; Rha is rhamnose; Man is mannose; X may be Rha or Man; Z isattached to Glc residue 2 and may be α-L-Rha-(1-4)-α-L-Rha, α-L-Man orα-L-Rha; W is attached to Glc residue number 1 and may beβ-D-Glc-(1-6)-α-D-Glc, β-D-Glc-(1-6)-β-D-Glc or α-L-Rha, subscripts vand y may be 0, 0.33, 0.5, 0.67 or 1, and wherein the “reducing end” ofthe polymer is toward the X residue of the backbone. As used herein, theterm “backbone” or “main chain” refers to that portion of the structurewhich excludes chains W and Z, i.e., when v and y are equal to 0.

Some members of the sphingan polysaccharide family are acetylated atvarious positions. However, the polysaccharides may be subjected tochemical deacylation in a conventional manner to remove the acyl groups.For example, gellan has the same carbohydrate backbone as welan (i.e.,X=Rha), but lacks the side chain sugar (i.e., v=0 and y=0) and theglucose residue 1 is fully substituted with glycerate. The gellansubunit structure is also partially acetylated at glucose residue 1.“Deacylated” as used herein means lacking glyceryl and acetyl groups.

As indicated above, one example of a sphingan exopolysaccharide isgellan gum. “Gellan gum” means a polysaccharide having the carbohydraterepeat structure —[(L)rhamnose-(D)glucose-(D)glucuronicacid-(D)glucose]—with glyceryl and acetyl groups attached to the glucoseresidue immediately to the reducing side of rhamnose. In standardpractice the reducing end of an oligosaccharide is placed at the right.Gellan gum has, on average, about one glyceryl group per repeat unit andabout one acetyl group per two repeat units.

As further described herein, parental strains of Shpingomonas producesphingan exopolysaccharide in the form of a capsule. As used herein“capsule” means a polysaccharide attached to the surface of theproducing bacterial cell, and which remains attached even after aqueousdilution, and which cannot be separated from the attached cells bysedimentation or centrifugation.

The term “Shpingomonas ” as used herein refers to strains ofgram-negative bacteria from the genus Shpingomonas which produceexopolysaccharides or sphingans, as described above. Thesphingan-producing family of gram-negative bacteria was first identifiedas belonging to the genus Shpingomonas in 1993 (See Pollock, J. Gen.Microb., 139, 1939 (1993)).

Shpingomonas bacteria useful in the present invention includeShpingomonas bacteria derived from parental strains that are geneticallymutated or which have undergone induced mutagenesis.

As used herein “parental strain” means the bacterial strain or anindividual bacteria before any treatment such as induced mutagenesis,which is intended to modify the genetic content or phenotype of aparental strain. This is meant to clearly distinguish a parental strainfrom a mutated or genetically-modified derivative strain that may beobtained from a parental strain or bacteria.

“Genetically mutated” as used herein means the quality of having beensubjected to either spontaneous or induced mutagenesis, and exhibitingproperties that distinguish the mutated bacteria or strain of bacteriafrom the parental strain.

“Induced mutagenesis” as used herein means the treatment of bacterialcells with agents commonly known to induce the formation of geneticmutations in DNA, including, but not limited to chemicals,electromagnetic radiation, biological agents such as viruses, plasmids,insertion elements or transposons.

The Shpingomonas bacteria of the present invention produceexopolysaccharides in the form of a slime. As used herein “slime” meansa polysaccharide which is not attached to the producing bacterial celland which can be substantially separated from cells by sedimentation orcentrifugation of the fermentation broth or after aqueous dilution ofthe broth, and in the absence of heat treatment, or other physical orchemical treatments of the broth. Bacteria which produce anexopolysaccharide in the form of a slime can be distinguished from thosethat produce a capsular polysaccharide by observation with a lightmicroscope. The encapsulated Shpingomonas bacteria form multicellularaggregates held together by the polysaccharide chains attached to thesurfaces of the cells, in contrast to the evenly dispersed slime-formingbacterial cells which have no capsule holding them together inaggregates.

The term “biosynthesis” as used herein describes the biologicalproduction or synthesis of sphingan by Shpingomonas bacteria. Sphinganexopolysaccharides are synthesized from individual carbohydrate units ina series of steps controlled by a number of enzymes of the bacteria.

The term “biomass” refers to the exopolysaccharide plus bacterial cellsin a bacterial culture.

Strain Development

The present invention utilized strains of Shpingomonas bacteria whichhave been genetically mutated to synthesize and secrete sphinganexopolysaccharides in a slime form. In this aspect of the invention,parental Shpingomonas strains which produce sphingan exopolysaccharidein a capsule form were subjected to procedures to provide geneticallymutated Shpingomonas bacteria. Examples of parental strains utilizedincluded S60 (ATCC 31461), S130 (ATCC 31555), S88 (ATCC 31554) and S7(ATCC 53159). Examples of genetically mutated Shpingomonas bacteriauseful in the present invention include ATCC PTA-3487 (strain X287)which produces gellan gum in a slime form, ATCC PTA-3486 (strain X530)which produces welan gum in a slime form, ATCC PTA-3485 (strain Z473)which produces exopolysaccharide S-88 in a slime form, and ATCC PTA-3488(strain X031) which produces exopolysaccharide S-7 in a slime form.

Fermentation

Another aspect of the present invention relates to the enhancedproduction of sphingan exopolysaccharide. To produce sphinganexopolysaccharide, genetically mutated Shpingomonas bacteria arecultured under suitable fermentation conditions, which are well known inthe art and which are generally described in U.S. Pat. No. 5,854,034which is hereby incorporated by reference. To summarize, a suitablemedium for culturing the genetically mutated Shpingomonas bacteria is anaqueous medium which generally contains a source of carbon such as, forexample, carbohydrates including glucose, lactose, sucrose, maltose ormaltodextrins, a nitrogen source such as, for example, inorganicammonium, inorganic nitrate, organic amino acids or proteinaceousmaterials such as hydrolyzed yeast, soy flour or casein, distiller'ssolubles or corn steep liquor, inorganic salts and vitamins. A widevariety of fermentation media will support the production of sphingansaccording to the present invention.

The carbohydrates are included in the fermentation broth in varyingamounts but usually between about 1% and 5% by weight of thefermentation medium. The carbohydrates may be added all at once prior tofermentation or alternatively, during fermentation. The amount ofnitrogen may range from about 0.01% to about 0.2% by weight of theaqueous medium. A single carbon source or nitrogen source may be used,as well as mixtures of these sources.

Among the inorganic salts which find use in fermenting Shpingomonasbacteria are salts which contain sodium, potassium, ammonium, nitrate,calcium, phosphate, sulfate, chloride, carbonate and similar ions. Tracemetals such as magnesium, manganese, cobalt, iron, zinc, copper,molybdenum, iodide and borate may also be advantageously included.Vitamins such as biotin, folate, lipoate, niacinamide, pantothenate,pyridoxine, riboflavin, thiamin and vitamin B₁₂ and mixtures thereof mayalso be advantageously employed.

The fermentation is carried out at temperatures between about 25° and35° C., with optimum productivity obtained within a temperature range ofabout 28° C. and 32° C. The inoculum is prepared by standard methods ofvolume scale-up, including shake flask cultures and small-scalesubmerged stirred fermentation. The medium for preparing the inoculumcan be the same as the production medium or can be any one of severalstandard media well-known in the art, such as Luria broth or YM medium.The concentration of carbohydrate can be reduced in the seed cultures toless than about 1% by weight. More than one seed stage may be used toobtain the desired volume for inoculation. Typical inoculation volumesrange from about 0.5% to about 10% of the total final fermentationvolume.

The fermentation vessel typically contains an agitator to stir thecontents. The vessel also may have automatic pH and foaming controls.The production medium is added to the vessel and sterilized in place byheating. Alternatively, the carbohydrate or carbon source may besterilized separately before addition. A previously grown seed cultureis added to the cooled medium (generally, at the fermentationtemperature of about 28° to about 32° C.) and the stirred culture isfermented for about 48 to about 96 hours, producing a broth having aviscosity of from about 15,000 to about 20,000 cp and from about 10 toabout 15 g/L sphingan exopolysaccharide in the slime form. Thefermentation of a corresponding parental Shpingomonas bacteria willtypically provide a broth having a viscosity of from about 25,000 toabout 50,000 cp.

In this aspect, the invention provides an exopolysaccharide in slimeform obtained from Shpingomonas bacteria grown in submerged, stirred andaerated liquid culture. The concentration of dissolved oxygen in theliquid culture exceeds about 5% of saturation of water after 24 hours ofculturing. Similar fermentations with parental strains resulted in 0%dissolved oxygen after 24 hours. The lower viscosity provided by theexopolysaccharide in slime form results in improved aeration whichallows the Shpingomonas bacteria to be productive in culture for alonger period of time.

In another aspect of the present invention, fermentation may be carriedout in a semi-batch process where bacteria from one fermentation areused as an inoculum for a subsequent fermentation. In this aspect,Shpingomonas bacteria which have been separated from theexopolysaccharides which they produced may be added to a freshfermentation broth, or a fresh fermentation broth may be added to theremaining Shpingomonas bacteria. Hence, this aspect of the inventionprecludes the need to provide a separate seed culture.

Processing of Fermentation Broth

Several approaches may be utilized to further process the fermenationbroth for recovery of exopolysaccharides. For example,exopolysaccharides may be directly precipitated from the fermentationbroth, the fermentation broth may be first clarified and thenprecipitated, the fermentation broth may be deacylated followed byprecipitation, or the fermentation broth may be deacylated followed byclarification and precipitation.

In one aspect of the invention, a fermentation broth having at leastabout 1% w/v amount of exopolysaccharides and a viscosity of not morethan about 25,000 cp is further processed for recovery ofexopolysaccharides. In this aspect of the invention, from about 1 toabout 1.5 volumes of alcohol is added directed to the fermentation brothat a temperature of about 25° C. to about 50° C. Alternatively, afermentation broth may be first deacylated and then clarified prior toaddition of alcohol as described in U.S. Pat. No. 4,326,052, which ishereby incorporated by reference. Alcohol effective for theprecipitation of exopolysaccharides include ethanol, isopropylalcohol,propanol, butanol, and mixtures thereof. Precipitation may be in batchmode or semi-continuous or continuous mode depending on the type ofmixing device used. If mixing is impeded by high viscosity then adilution with water is used before adding alcohol.

After fermentation, exopolysaccharides are then separated from cellulardebris with sedimentation or filtration. Sedimentation may beaccomplished by centrifugation. In this aspect of the invention, afterprecipitation, any of several methods known in the art for recovering,pressing, drying and milling to give a homogenous exopolysaccharidepowder can be used.

In accordance with this aspect of the invention, the process yieldsabout 1 to about 2% (w/v, based on the original broth volume)exopolysaccharide. The purity typically exceeds at least about 80%.

The following examples illustrate methods for carrying out the inventionand should be understood to be illustrative of, but not limiting upon,the scope of the invention which is defined in the appended claims.

EXAMPLES Example I

Gellan gum was produced from the Shpingomonas mutant strain X287, whichis derived from the wild type strain ATCC31461, and from the ATCC31461parent strain by aerated, stirred, submerged fermentation. Strain X287was obtained by a reproducible multi-step regimen of induced mutagenesisand selection of preferred properties.

The growth medium contained (per liter of deionized water): 1 g ammoniumnitrate, 0.5 g soluble hydrolyzed soy protein (Soy Peptone from Marcor),3.2 g dibasic potassium phosphate, 1.6 g monobasic potassium phosphate,0.1 g magnesium sulfate (heptahydrate), trace minerals, and glucose. Thetrace minerals contained (per liter of final medium): 2.7 mg FeCl₃-6H₂O,1.36 mg ZnCl₂, 1.98 mg MnCl₂-4H₂O, 240 μg CoCl₂-6H₂O, 240 μgNa₂MoO₄-2H₂O, and 250 μg CuSO₄-5H₂O. The final fermentation mediumcontained 25 g glucose per liter, and the preculture medium contained 10g glucose per liter.

A vial of frozen cells was thawed and added to a preculture of 500 ml ina baffled flask and incubated at 30° C. on a rotary shaker for about16-20 hours or until the cell density measured by the absorbance at 600nm was about 3-5. The frozen cells were samples taken from a previouspreculture flask which had been inoculated with a single colony of thebacterial strain grown on an agar plate containing the same medium.

An amount of a preculture was added to the fermentor to give aninoculation of about 5-10% by volume. The fermentor was either a NewBrunswick model III or model 3000 and contained 4 liter of medium. Thefermentor was aerated with 1 volume of air per volume of fermentationmedium, and was maintained at 30° C. The agitation speed was controlledby the amount of dissolved oxygen which was set to a minimum of 30% ofsaturation. During the fermentations the agitation speed reached themaximum of 1000 rpm and this was followed by a decrease in the level ofdissolved oxygen to between 0-30%.

The following table shows the results of submerged fermentations withthe parent strain ATCC31461 and the slime-forming mutant strain X287.For the X287 mutant culture where the polysaccharide is not attached tothe cells, the reduced viscosity results in more efficient mixing andaeration, and lower energy consumption.

IPA Glucose Duration precipitate consumed Viscosity Strain (hr) A600(g/l) (g/l) (cp at 12 rpm) ATCC31461 27 16.7 12.8 22.7 21700 X287 3215.7 15.1 24.4 12900

During product recovery by alcohol precipitation two volumes ofisopropylalcohol are required to precipitate gellan gum made by the wildtype strain ATCC31461. In contrast the gellan gum made in the form of aslime by strain X287 is precipitated from the broth with only one volumeof isopropylalcohol. In addition, the gellan gum made in the form of aslime can be precipitated with isopropylalcohol at 25-30° C. while thewild type gellan requires a heating step before adding the alcohol.

Example II

Welan gum was produced in slime form from any of several mutant strains,of which strains X530 and X319 are representative, which were obtainedby multi-step regimen applied to the wild type parent strain ATCC31555,consisting of induced or spontaneous mutagenesis and selection ofpreferred properties.

Bacterial strain X530 was grown in liquid medium (described in ExampleI) in shaking flasks. Strain X530 synthesized welan gum which wasunattached to the cells, in the form of a slime. The appearance of theculture was observed with a light microscope. Whereas the parental wildtype strain formed multicellular aggregates held together by thecapsular polysaccharides attached to each cell, the cells of strain X530did not form aggregates and were free to distribute evenly throughoutthe culture medium as single cells. The following measurements were madeand are tabulated below: culture viscosity (centipoise at 12 rpm withspindle #4 for a Brookfield LVTDV-II viscometer), cell density(absorbance at 600 nm), and weight of the biomass (polysaccharide pluscells) precipitated directly from the culture broth at 25° C. with 2volumes of isopropylalcohol (g/l).

Cell Density Culture Precipitated Strain A600 Viscosity cp Biomass g/lATCC 31555 14.7 6610 7.8 ATCC 31555 14.8 8300 8.3 X530 slime 14.7 59109.8 X319 slime 17.7 4960 8.7

In addition, the precipitations were performed at 90° C. and theappearance of the precipitated polysaccharides in the alcohol-brothmixtures was recorded. Addition of 2 volumes of isopropylalcohol to theculture of strain X530 caused the formation of cohesive clots at either25° C. or 90° C., while in contrast the precipitated polysaccharides forthe wild type parent strain formed dispersed fragmented clots at 25° C.and cohesive clots at 90° C.

Example III

Polysaccharide S-88 was produced in slime form from any of severalmutant strains, of which strains X099 and Z473 are representative, whichwere obtained by a multi-step regimen applied to the wild type parentstrain ATCC31554, consisting of induced or spontaneous mutagenesis andselection of preferred properties.

The growth medium contained (per liter of water): 1 g ammonium nitrate,0.5 g soluble hydrolyzed soy protein (Soy Peptone from Marcor), 3.2 gdibasic potassium phosphate, 1.6 g monobasic potassium phosphate, 0.2 gmagnesium sulfate (heptahydrate), trace minerals, and glucose. The traceminerals contained (per liter of final medium): 2.7 mg FeCl₃-6H₂O, 1.36mg ZnCl₂, 1.98 mg MnCl₂-4H₂O, 240 μg CoCl₂-6H₂O, 240 μg Na₂MoO₄-2H₂O,and 250 μg CuSO₄-5H₂O. The final fermentation medium contained 30 gglucose per liter, and the preculture medium contained 20 g glucose perliter.

A vial of frozen cells was thawed and added to a preculture of 500 ml ina baffled flask and incubated at 30° C. on a rotary shaker for about16-20 hours or until the cell density measured by the absorbance at 600nm was about 3-5. The frozen cells were samples taken from a previouspreculture flask which had been inoculated with a single colony of thebacterial strain grown on an agar plate containing the same medium.

An amount of a preculture was added to the fermentor to give aninoculation of about 5-10% by volume. The fermentor was a New Brunswickmodel III and contained 4 liter of medium. The fermentor was aeratedwith 1 volume of air per volume of fermentation medium, and wasmaintained at 30° C. The agitation speed was controlled by the amount ofdissolved oxygen which was set to a minimum of 30% of saturation. Duringthe fermentations the agitation speed reached the maximum of 1000 rpmand this was followed by a decrease in the level of dissolved oxygen tobetween 0-30%.

The following table shows the results of submerged fermentations withthe parent strain ATCC31554 and the slime-forming mutant strains X099and Z473. For the X099 and Z473 mutant cultures where the polysaccharideis not attached to the cells, the productivity is increased, asindicated by the increased viscosity of the broth and the increasedweight of the IPA precipitate. The polysaccharide precipitated with onlyone volume of isopropylalcohol per volume of culture broth, in contrastto two volumes for the wild type parent ATCC31554. The cells of strainsX099 and Z473 can be removed from the polysaccharide by slow speedcentrifugation (about 5000×G), which was not possible with the ATCC31554wild type parent strain.

IPA Glucose Duration precipitate consumed Viscosity Strain (hr) A600(g/l) (g/l) (cp at 12 rpm) ATCC31554 36 14.0 11.6 21.9 8600 X099 36 15.815.0 23.1 14300 Z473 36 15.7 12.0 20.8 13100

Example IV

Polysaccharide S-7 was produced in slime form from a mutant strain X031and in capsule form by ATCC21423 when cultured by fermentation. StrainX031 is a spontaneous mutant derived from the ATCC 21423 strain. Thegrowth medium contained (per liter of water) 1 g ammonium nitrate, 0.5 gsoluble hydrolyzed soy protein (Soy Peptone from Marcor), 3.2 g dibasicpotassium phosphate, 1.6 g monobasic potassium phosphate, 0.2 gmagnesium sulfate (heptahydrate), trace minerals, and glucose. The traceminerals contained (per liter of final medium): 2.7 mg FeCl₃-6H₂O, 1.36mg ZnCl₂, 1.98 mg MnCl₂-4H₂O, 240 μg CoCl₂-6H₂O, 240 μg Na₂MoO₄-2H₂O,and 250 μg CuSO₄-5H₂O. The final fermentation medium contained 30 gglucose per liter, and the preculture medium contained 20 g glucose perliter.

A vial of frozen cells was thawed and added to a preculture of 500 ml ina baffled flask and incubated at 30° C. on a rotary shaker for about16-20 hours or until the cell density measured by the absorbance at 600nm was about 3-5. The frozen cells were samples taken from a previouspreculture flask which had been inoculated with a single colony of thebacterial strain grown on an agar plate containing the same medium.

An amount of a preculture was added to the fermentor to give aninoculation of about 5-10% by volume. The fermentor was a New Brunswickmodel III and contained 4 liter of medium. The fermentor was aeratedwith 1 volume of air per volume of fermentation medium, and wasmaintained at 30° C. The agitation speed was controlled by the amount ofdissolved oxygen which was set to a minimum of 30% of saturation. Duringthe fermentations the agitation speed reached the maximum of 1000 rpmand this was followed by a decrease in the level of dissolved oxygen tobetween 0-30%.

The following table shows the results of submerged fermentations withthe parent strain ATCC21423 and the slime-forming mutant strain X031.For the X031 mutant culture where the polysaccharide is not attached tothe cells, the reduced viscosity results in more efficient mixing andaeration, and lower energy consumption. The cells of strain X031 couldbe removed from the polysaccharide by slow speed centrifugation (about5000×G), which was not possible with the ATCC21423 wild type parentstrain. The cells of the wild type strain ATCC21423 were only removed byslow speed centrifugation after partial hydrolysis of the polysaccharidewith elevated temperature (about 90-121° C.) with the pH in the acidicrange of 5-7. By breaking the polysaccharide chains at internalpositions in the polymers the cells and a fraction of the polysaccharidebecame disconnected.

IPA Glucose Duration precipitate consumed Viscosity Strain (hr) A600(g/l) (g/l) (cp at 12 rpm) ATCC21423 48 8.5 17.0 26.0 31100 X031 48 9.617.7 30.5 20400

Example V

Shpingomonas mutant strain X287 and its parental strain ATCC31461 werecultured by submerged aerated liquid fermentation as in Example I. Thefollowing table shows the time course of changes in viscosity anddissolved oxygen during the duration of the fermentation.

Duration Dissolved Viscosity Strain (Hrs) Oxygen (%) (cp) ATCC31461 1229 250 24 0 18,000 48 0 26,400 72 0 29,700 X287 12 36 725 24 28 6,460 4810 11,000 72 0 14,200

Numerous modifications and variations in practice of the invention areexpected to occur to those skilled in the art upon consideration of theforegoing detailed description of the invention. Consequently, suchmodifications and variations are intended to be included within thescope of the following claims.

1. A fermentation broth comprising slime-forming Sphingomonas bacterialcells selected from the group consisting of ATCC PTA-3487, ATCCPTA-3486, ATCC PTA-3485, ATCC PTA-3488, and mixtures thereof and anexopolysaccharide in slime form that is obtained by a processcomprising: culturing a Sphingomonas bacteria selected from the groupconsisting of ATCC PTA-3487, ATCC PTA-3486, ATCC PTA-3485, ATCCPTA-3488, and mixtures thereof for a time and temperature effective forproviding a broth from which a sphingan exopolysaccharide can berecovered with an alcohol precipitation method at a temperature of about25° C. to about 50° C. wherein the method yields at least about 10 gramsof sphingan exopolysaccharide per liter of broth.
 2. A fermentationbroth according to claim 1 wherein fermentation is conducted from about48 to about 96 hours at a temperature of about 25° C. to about 35° C. 3.A fermentation broth according to claim 1 wherein in the alcoholprecipitation method from about 1 to about 1.5 volumes of alcohol areadded to the fermentation broth.
 4. A fermentation broth comprisingslime-forming Sphingomonas bacterial cells and an exopolysaccharide in aslime form, wherein the exopolysaccharide can be recovered from saidfermentation broth by alcohol precipitation at a temperature of about25° C. to about 50° C. to yield at least about 10 grams ofexopolysaccharide per liter of broth, and wherein the Sphingomonas isselected from the group consisting of ATCC PTA3487, ATCC PTA-3486, ATTCPTA-3485, ATCC PPA-3488, and mixtures thereof.
 5. A fermentation brothaccording to claim 4 wherein the exopolysaccharide in slime form is asphingan exopolysaccharide having the general formula

wherein Glc is glucose, GlcA is glucuronic acid or 2-deoxy-glucuronicacid, Rha is rhamnose, Man is mannose, X is Rha or Man, Z is attached toGlc residue 2 and is α-L-Rha-(1-4) -α-L-Rha, α-L-Man or α-L-Rha, W isattached to Glc residue number 1 and is β-D-Glc-(1-6) -α-D-Glc,β-D-Glc-(1-6)-β-D-Glc or α-L-Rha, subscripts v and y are 0, 0.33, 0.5,0.67 or
 1. 6. A fermentation broth comprising slime-forming Sphingomonasbacterial cells and an exopolysaccharide in a slime form having thegeneral formula

wherein Glc is glucose, GlcA is glucuronic acid or 2-deoxy-glucuronicacid, Rha is rhamnose, Man is mannose, X is Rha or Man, Z is attached toGlc residue 2 and is α-L-Rha-(1-4)-α-L-Rha, α-L-Man or α-L-Rha, W isattached to Glc residue number 1 and is β-D-Glc-(1-6)-α-D-Glc,β-D-Glc-(1-6)-β-D-Glc or α-L-Rha, subscripts v and y are 0, 0.33, 0.5,0.67 or 1, wherein the fermentation broth has at least about 1% w/vexopolysaccharide and a viscosity of not more than about 25,000 cp, andwherein the Sphingomonas is selected from the group consisting of ATCCPTA-3487, ATCC PTA3486, ATCC PTA-3485, ATCC PTA-3488, and mixturesthereof.
 7. A fermentation broth comprising slime-forming Shpingomonasbacteria selected from the group consisting of ATCC PTA-3487, ATCCPTA-3486, ATCC PTA-3485, ATCC PTA-3488, and mixtures thereof and anexopolysaccharide in a slime form produced by the Shpingomonas bacteria,wherein the Shpingomonas bacteria are grown in a submerged, aeratedliquid culture, wherein a concentration of dissolved oxygen exceedsabout 5% of saturation of water after 24 hours of culturing, and whereinthe exopolysaccharide in slime form is produced by Shpingomonas selectedfrom the group consisting of ATCC PTA-3487, ATCC PTA-3486, ATCCPTA-3485, ATCC PTA-3488, and mixtures thereof.
 8. A fermentation brothaccording to claim 7 wherein the exopolysaccharide in slime form is aSphingomonas exopolysaccharide having the general formula

wherein Glc is glucose, GlcA is glucuronic acid or 2-deoxy-glucuronicacid, Rha is Rhamnose, Man is mannose, X is Rha or Man, Z is attached toGlc residue 2 and is α-L-Rha-(1-4) -α-L-Rha, α-L-Man or α-L-Rha, W isattached to Glc residue number 1 and is β-D-Glc-(1-6) -α-D-Glc,βD-Glc-(1-6)-β-D-Glc or α-L-Rha, subscripts v and y are 0, 0.33, 0.5,0.67 or
 1. 9. The fermentation broth of claim 4 wherein theexopolysaccharide is selected from the group consisting of gellan,welan, rhamsan, S-88, S-7, S-198, NW-11 and S-657.
 10. The fermentationbroth of claim 9 wherein the exopolysaccharide is selected from thegroup consisting of gellan, welan, S-7 and S-88.